Bottom Line:
PPARγ is a key regulator of glucose homeostasis and insulin sensitization.PPARγ must heterodimerize with its dimeric partner, the retinoid X receptor (RXR), to bind DNA and associated coactivators such as p160 family members or PGC-1α to regulate gene networks.The solution structures reveal an asymmetry of the overall structure due to the crucial role of the DNA in positioning the heterodimer and indicate asymmetrical binding of TIF2 to the heterodimer.

ABSTRACTPPARγ is a key regulator of glucose homeostasis and insulin sensitization. PPARγ must heterodimerize with its dimeric partner, the retinoid X receptor (RXR), to bind DNA and associated coactivators such as p160 family members or PGC-1α to regulate gene networks. To understand how coactivators are recognized by the functional heterodimer PPARγ/RXRα and to determine the topological organization of the complexes, we performed a structural study using small angle X-ray scattering of PPARγ/RXRα in complex with DNA from regulated gene and the TIF2 receptor interacting domain (RID). The solution structures reveal an asymmetry of the overall structure due to the crucial role of the DNA in positioning the heterodimer and indicate asymmetrical binding of TIF2 to the heterodimer.

fig4: Solution structure of PPARγ2/RXRα bound to Cyp4A1 PPRE. (a) Scattering profiles of PPARγ2ΔNTD/RXRαΔNTD/PPRE. Experimental data are shown as red dots. Green fit is computed from the solution structure of the complex. (b) Distance distribution function computed from the X-ray scattering pattern using the program GNOM. (c) Most typical molecular envelope of PPARγ2ΔNTD/RXRαΔNTD/PPRE generated by DAMMIF (beads model shown as a grey surface) together with the refined model by rigid body refinement using the program SASREF (fit to the experimental data with χR = 0.98). (d) Pseudoatomic solution structure of PPARγ2ΔNTD/RXRαΔNTD/Cyp4A1 PPRE shown in schematic cartoon representation together with the sequence of the DNA.

Mentions:
PPARγ/RXRα without DNA has been shown to be elongated with no interdomain interactions [17]. In complex with idealized DNA, the first atomic resolution structure of integral PPARγ/RXRα obtained by X-ray crystallography [15] confirmed the structural information concerning isolated domains [11, 31, 32] and also revealed an interdomain contact between the LBD of PPARγ and the DBD of RXR [13], although the functional correlation is limited to a single point mutation. The N-terminal domain, unfolded, was not visible in the electron density map. We previously measured the SAXS profile of PPARγ2/RXRα bound to an idealized DR1 [16] and have shown that the conformation in solution is different to that observed in the crystal structure with the heterodimer exhibiting an extended asymmetric shape without additional interdomain contacts between the DBDs and LBDs beyond the connection through the hinge regions. We have now characterized the solution structure of PPARγ2/RXRα full length or truncated of their NTDs and bound to a natural PPRE from CYP4A1. The structural parameters calculated from the experimental scattering patterns (Figures 4(a) and 4(b)) given in Table 1 reveal an extended shape of the complex. With the full length PPARγ complex, the structural parameters are even larger (Rg = 44 Å and Dmax⁡ = 160 Å), suggesting a distinct dissociated NTD. Low resolution models were reconstructed ab initio from the corresponding experimental scattering patterns. The most typical ab initio model of PPARγ2ΔNTD/RXRαΔNTD/CYP4A1 PPRE presented in Figure 4(c) clearly displays separate DBD and LDB domains similar to the architecture observed for the complex with the idealized DR1 and those observed for other NR heterodimers. For PPARγ2ΔNTD/RXRαΔNTD/PPRE, its atomic structure was refined against SAXS experimental data (Figure 4(a)). The position of the domains was adjusted by rigid body modeling using the available high resolution crystal structure of the complex (PDB ID: 3DZY). The model obtained by rigid body refinement agrees well with the ab initio models as seen from the superposition in Figure 4(c). The refined structure reveals an asymmetric shape with the LBD dimer positioned at the 5′ end of the DNA (Figure 4(d)), an asymmetry already observed for other heterodimers studied by SAXS [16].

fig4: Solution structure of PPARγ2/RXRα bound to Cyp4A1 PPRE. (a) Scattering profiles of PPARγ2ΔNTD/RXRαΔNTD/PPRE. Experimental data are shown as red dots. Green fit is computed from the solution structure of the complex. (b) Distance distribution function computed from the X-ray scattering pattern using the program GNOM. (c) Most typical molecular envelope of PPARγ2ΔNTD/RXRαΔNTD/PPRE generated by DAMMIF (beads model shown as a grey surface) together with the refined model by rigid body refinement using the program SASREF (fit to the experimental data with χR = 0.98). (d) Pseudoatomic solution structure of PPARγ2ΔNTD/RXRαΔNTD/Cyp4A1 PPRE shown in schematic cartoon representation together with the sequence of the DNA.

Mentions:
PPARγ/RXRα without DNA has been shown to be elongated with no interdomain interactions [17]. In complex with idealized DNA, the first atomic resolution structure of integral PPARγ/RXRα obtained by X-ray crystallography [15] confirmed the structural information concerning isolated domains [11, 31, 32] and also revealed an interdomain contact between the LBD of PPARγ and the DBD of RXR [13], although the functional correlation is limited to a single point mutation. The N-terminal domain, unfolded, was not visible in the electron density map. We previously measured the SAXS profile of PPARγ2/RXRα bound to an idealized DR1 [16] and have shown that the conformation in solution is different to that observed in the crystal structure with the heterodimer exhibiting an extended asymmetric shape without additional interdomain contacts between the DBDs and LBDs beyond the connection through the hinge regions. We have now characterized the solution structure of PPARγ2/RXRα full length or truncated of their NTDs and bound to a natural PPRE from CYP4A1. The structural parameters calculated from the experimental scattering patterns (Figures 4(a) and 4(b)) given in Table 1 reveal an extended shape of the complex. With the full length PPARγ complex, the structural parameters are even larger (Rg = 44 Å and Dmax⁡ = 160 Å), suggesting a distinct dissociated NTD. Low resolution models were reconstructed ab initio from the corresponding experimental scattering patterns. The most typical ab initio model of PPARγ2ΔNTD/RXRαΔNTD/CYP4A1 PPRE presented in Figure 4(c) clearly displays separate DBD and LDB domains similar to the architecture observed for the complex with the idealized DR1 and those observed for other NR heterodimers. For PPARγ2ΔNTD/RXRαΔNTD/PPRE, its atomic structure was refined against SAXS experimental data (Figure 4(a)). The position of the domains was adjusted by rigid body modeling using the available high resolution crystal structure of the complex (PDB ID: 3DZY). The model obtained by rigid body refinement agrees well with the ab initio models as seen from the superposition in Figure 4(c). The refined structure reveals an asymmetric shape with the LBD dimer positioned at the 5′ end of the DNA (Figure 4(d)), an asymmetry already observed for other heterodimers studied by SAXS [16].

Bottom Line:
PPARγ is a key regulator of glucose homeostasis and insulin sensitization.PPARγ must heterodimerize with its dimeric partner, the retinoid X receptor (RXR), to bind DNA and associated coactivators such as p160 family members or PGC-1α to regulate gene networks.The solution structures reveal an asymmetry of the overall structure due to the crucial role of the DNA in positioning the heterodimer and indicate asymmetrical binding of TIF2 to the heterodimer.

ABSTRACTPPARγ is a key regulator of glucose homeostasis and insulin sensitization. PPARγ must heterodimerize with its dimeric partner, the retinoid X receptor (RXR), to bind DNA and associated coactivators such as p160 family members or PGC-1α to regulate gene networks. To understand how coactivators are recognized by the functional heterodimer PPARγ/RXRα and to determine the topological organization of the complexes, we performed a structural study using small angle X-ray scattering of PPARγ/RXRα in complex with DNA from regulated gene and the TIF2 receptor interacting domain (RID). The solution structures reveal an asymmetry of the overall structure due to the crucial role of the DNA in positioning the heterodimer and indicate asymmetrical binding of TIF2 to the heterodimer.